Use of mono-3,6-anhydrocyclodextrins for solubilizing a hydrophobic compound and for controlling the purity of an enantiomer and process for the preparation of these cyclodextrins

ABSTRACT

The invention relates to the use of mono-3,6-anhydrocyclodextrins for solubilizing a hydrophobic compound and for controlling the purity of an enantiomer. 
     The mono-3,6-anhydrocyclodextrin complies with the following formula: ##STR1## in which n is equal to 5, 6 or 7. Preferably n is equal to 6. The hydrophobic compound can be a steroid such as prednisolone.

The present invention relates to the use of derivatives of cyclodextrinsfor solubilizing in an aqueous medium hydrophobic chemical compounds, inparticular pharmaceutically active molecules, by the inclusion of thesemolecules in the cyclodextrin derivative.

Cyclodextrins or cyclomaltooligosaccharides are compounds having anatural origin forming by a linking of 6, 7 or 8 glucose units bonded inχ-1,4. Numerous works have shown that these cyclodextrins could forminclusion complexes with hydrophobic molecules and thus permit thesolubilization of these molecules in aqueous media. Numerousapplications have been proposed for taking advantage of this phenomenon,particularly in the pharmaceutical field, as is described by D. Duchenein the work entitled "Cyclodextrins and their industrial uses", chapter6, pp 213 to 257, Editions de Sante, 1987. Pharmaceutical compositionsusing these cyclodextrins have also been marketed in Japan and Italy andmore recently in France, e.g. by Pierre Fabre Medicament forBrexin.sup.(R), which is an inclusion complex of Piroxicam inβ-cyclodextrin.

Among the usable cyclodextrins, β-cyclodextrin, which has 7 glucoseunits, is the most appropriate with regards to the size of its cavityand is the least expensive of the three, but its use causes certainproblems, because it is less soluble than other cyclodextrins and has ahemolytic character.

Consideration has also been given to the improvement of the propertiesof β-cyclodextrin by chemically modifying to make it more suitable.Several solutions have been envisaged and have led to the use of methylderivatives or hydroxyalkyl derivatives.

Methyl derivatives are much more soluble than the original cyclodextrinand they have good properties of solubilizing hydrophobic, organiccompounds, particularly in the case of 2,6-dimethyl-β-cyclodextrin.However, these methyl derivatives, apart from the fact that they aredifficult to obtain in the pure state, are unusable for pharmaceuticalapplications, particularly for injectable forms, due to their verypronounced hemolytic character.

The hydroxyalkyl derivatives more particularly developed by Janssen,e.g. hydroxypropyl-cyclodextrins have a very high solubility in waterand are only slightly hemolytic. However, their use remains difficultdue to their extreme chemical heterogeneity. In addition, substitutionscan limit the formation of inclusion complexes by steric hindrance andas yet no pharmaceutical application has been developed with thesederivatives.

The present invention specifically relates to the use of otherderivatives of cyclodextrins for the solubilization of hydrophobicchemical compounds making it possible to obviate these disadvantages. Italso relates to a novel process for the preparation of the cyclodextrinderivatives leading to very pure products without requiring laboriouspurification stages.

According to the invention, the process for solubilizing a hydrophobicchemical compound in an aqueous medium consists of combining thehydrophobic chemical compound with a mono-3,6-anhydrocyclodextrin offormula: ##STR2## in which n is equal to 5, 6 or 7, to for therewith awater-soluble inclusion complex.

The use in this process of the monoanhydrocyclodextrin derivativecomplying with the aforementioned formula has the advantage of improvingthe solubility, stability and bioavailability, in various administrationforms of the hydrophobic compound, particularly in the case ofpharmaceutically active molecules.

In particular, the solubility in water of these derivatives isconsiderable and greatly superior to that of the parent cyclodextrin,particularly with respect to β-cyclodextrin. Moreover, thesecyclodextrin derivatives have a much weaker hemolytic character than theparent cyclodextrin. These cyclodextrin derivatives also have theproperty of different affinities for the isomers of a mixture of isomersand can be consequently used in chromatography-based isomer separationprocesses. They also make it possible to easily carry out a puritycontrol of the enantiomer, because the inclusion complexes formed withracemic chiral compounds have in nuclear magnetic resonance spectrometryan easily observable separation of the characteristic signals of eachenantiomer.

According to the invention, preference is given to the use of themonoanhydro derivative of β-cyclodextrin, i.e. the derivative of formula(I) with n=6. However, it is also possible to use the derivatives ofχ-cyclodextrin (n=5) or γ-cyclodextrin (n=7).

The hydrophobic chemical compounds which can be solubilized in aqueousmedia by means of these cyclodextrins can be of different types.

As examples of such compounds, reference can be made to cosmeticproducts, vitamins, pharmaceutically active molecules like thosedescribed by D. Duchene in the work entitled "Cyclodextrins and theirindustrial uses", chapter 6, pp 213-257, Editions de Sante, 1987.

Preferably, in the present invention, the hydrophobic chemical compoundis a pharmaceutically active molecule.

As examples of such molecules, reference can be made to steroids, e.g.prednisolone, anit-epileptic agents such as carbamazepine, andanti-cancer agents.

The cyclodextrin derivatives of formula (I) used in the invention can beprepared by the process described in Chemistry Letters, pp 543-546,1988, by reacting a corresponding monotosyl derivative with an aqueoussoda solution. After this reaction, it is possible to isolate themonoanhydro derivative in the pure state by carrying out advanced highperformance liquid chromatography purifications in order to eliminatethe byproducts and salts.

In addition, according to the invention, use is preferably made for thepreparation of these derivatives of a simpler process making it possibleto avoid these purification stages.

The invention also relates to a process for the preparation of amono-3,6-anhydrocyclodextrin of formula: ##STR3## in which n is equal to5, 6 or 7 consisting of reacting a monotosyl derivative of cyclodextrinof formula: ##STR4## in which R is the tosyl group and n is equal to 5,6 or 7, with lithia in an aqueous medium.

The use of lithia in place of soda makes it possible to much more easilyobtain the monoanhydrocyclodextrin derivative and to then carry out itsseparation from the reaction medium under better conditions.

This separation can be carried out by precipitation after theacidification of the reaction medium and by repeating theseprecipitation stages, it is possible to isolate the cyclodextrinderivative in the pure state without it being necessary to carry outcomplimentary, chromatography-based purification stages.

Precipitation can be carried out by adding the aqueous medium afteracidification to an organic solvent, such as acetone, and thenseparating the precipitate formed by centrifuging. This makes itpossible to eliminate all the sulphonic acid derivatives soluble in theorganic solvent, as well as the salts, because lithium chloride issoluble in an organic solvent such as acetone.

The invention also relates to the inclusion complexes of amono-3,6-anhydrocyclodextrin complying with formula (I) with ahydrophobic chemical compound, particularly a pharmaceutically activemolecule.

This inclusion complex can be prepared by conventional processes, e.g.by adding to a solution or suspension of themono-3,6-anhydrocyclodextrin used, a solution of the hydrophobiccompound in an appropriate organic solvent, e.g. acetone. It is thenpossible to isolate the inclusion complex formed by lyophilization.

These inclusion complexes, when they are formed with pharmaceuticallyactive molecules, can in particular be used in pharmaceuticalcompositions, which also include the pharmaceutically acceptablevehicle.

These pharmaceutical compositions, which can be administered orally orparenterally, are e.g. solutions, powders, suspensions, etc. and inparticular injectable solutions.

As stated hereinbefore, the inclusion complexes formed with themono-3,6-anhydrocyclodextrins of formula (I), and racemic, chiralcompounds have the interesting property, in nuclear magnetic resonance,of separate signals for each enantiomer.

The invention also relates to a process for controlling the purity of anenantiomer of an organic compound, which consists of combining thisenantiomer with a mono-3,6-anhydrocyclodextrin according to formula (I)for forming an inclusion complex of said enantiomer and subjecting thecomplex obtained to nuclear magnetic resonance spectrometry fordetecting the possible presence of the other enantiomer on the spectrumobtained.

The invention also relates to a process for the separation of isomers bychromatography, which consists of circulating a mixture of these isomersin a column filled with a solid chromatographic support, to which iscovalently fixed a mono-3,6-anhydrocyclodextrin of formula (I) accordingto the invention and separately collecting the isomers at the columnexit.

The chromatographic support used can be formed by an insoluble polymeror microparticles of silica. The insoluble polymers can in particular beagarose and polysaccharides of the Sephadex.sup.(R) types.

The chemical grafting of the cyclodextrins according to the invention tothe chromatographic supports can be carried out by means of a couplingreagent such as epichlorohydrin, which ensures the coupling between anOH group of the cyclodextrin and an OH group of the chromatographicsupport.

The property of the cyclodextrins according to the invention of having adifferent affinity for each of the isomers makes it possible to obtain agood separation thereof by chromatography. The isomers can be opticalisomers, position isomers or diastereoisomers.

Other features and advantages of the invention can be better gatheredfrom the following illustrative and non-limitative examples, withreference to the attached drawings, wherein show:

FIG. 1 The nuclear magnetic resonance spectrum of an inclusion complexof mono-3,6-anhydrocyclomaltoheptaose with a racemic mixture ofdothiepin.

FIG. 2 A larger-scale portion of the spectrum of FIG. 1.

EXAMPLE 1 Preparation of mono-3,6-anhydrocyclomaltoheptaose

In order to carry out the synthesis according to the process of theinvention, preparation firstly takes place of themono-6-tosyl-deoxy-cyclomaltoheptaose by the action of p-toluenesulphonyl chloride on β-cyclodextrin in an aqueous medium.

60 g of cyclomaltoheptaose (52.8 mmole) are suspended in 500 ml ofdistilled water. Dropwise addition takes place of 6.57 g (164 mmole) ofcaustic soda dissolved in 20 ml of water over 5 minutes and with strongmagnetic stirring. To the clear solution obtained are added 10.08 g(52.9 mmole) of p-toluene sulphonyl chloride (tosyl chloride) in 30 mlof acetonitrile in dropwise manner over 10 minutes. After stirring for 2hours at ambient temperature, the precipitate formed is eliminated byfiltration and the filtrate is kept for 48 hours at 4° C. Theprecipitate is isolated by filtration in vacuo, washed with 50 ml of icewater and recrystallized immediately in boiling water. After one nightat 4° C., the precipitate is filtered and dried in vacuo at 30° C. Thisgives 7.5 g (12%) of a pure compound in accordance with thespecifications.

Dissolving then takes place of 100 mg of the previously obtained6-tosyl-6-deoxy-cyclomaltoheptaose in 10 ml of 1M LiOH in water,dissolving being immediate. The solution is kept at 40° C. for 15 hoursand is then acidified to a pH of approximately 3 using 1M HCl. Thesolution is then added dropwise and accompanied by stirring to 20 ml ofpure acetone. A vitreous precipitate is formed, which is isolated bycentrifuging (6000 rpm, 10 min) and the precipitate is redissolved in0.5 ml of water. This precipitation is repeated and the residue againisolated by centrifuging, followed by redissolving in water andlyophilization.

The structure of the product obtained is confirmed by nuclear magneticresonance of the high field proton and in this way spectralcharacteristics are obtained in accordance with those given in ChemistryLetters, pp 543-546, 1988.

The solubility in water of the compound obtained is 520 g/l at 25° C.,i.e. close to 30 times that of the original cyclodextrin, which is 18g/l. This solubility is also at least twice that of χ andγ-cyclodextrins.

The hemolytic properties of this derivative were tested by contacting0.4 ml of a suspension of human erythrocytes and a 5 mmole/l solution ofthis derivative, at a pH of 7.4, for 30 min at 37° C. Under theseconditions, the derivative reveals 0% hemolysis, whereas the hemolysispercentage is 50% for β-cyclodextrin under the same conditions.

Other tests revealed the absence of hemolytic properties at much higherconcentrations (no hemolysis detectable at 50 mmole/l).

EXAMPLE 2 Preparation of an inclusion complex ofmono-3,6-anhydrocyclo-maltoheptaose and prednisolone

The prednisolone is in accordance with the following formula: ##STR5##and has a very low solubility in water (0.25 mg/ml at 25° C., i.e. 0.7mole/l). 10 μmole of mono-3,6-anhydrocyclomaltoheptaose prepared inexample 1 are dissolved in 1 ml of pure water (apyrogenic water forinjections) and addition takes place of 5 μmole of prednisolone in theform of a 50 mmole/l concentrated solution in acetone. The acetone iseliminated under nitrogen bubbling and the solution lyophilized.

The residual solid, which contains 10 μmole of the cyclodextrinderivative and 5 μmole of prednisolone, is redissolved in the minimum ofwater at 25° C. This minimum corresponds to 50 μl of water, whichindicates a maximum prednisolone solubility in water of 100 mmole/l inthe presence of said cyclodextrin derivative at a concentration of 200mmole/l. Under the same conditions, β-cyclodextrin only makes itpossible to solubilize prednisolone at 9 mmole/l.

Thus, a much better result is obtained with the monoanhydro derivativeof said cyclodextrin.

EXAMPLE 3 Preparation of an inclusion complex ofmono-3,6-anhydrocyclo-maltoheptaose with dothiepin in the form of theracemic mixture

The dothiepin corresponds to the formula: ##STR6##

This molecule is optically active by the absence of symmetry withrespect to the aromatic plane and the commercial compound is a racemicproduct. The inclusion complex of this racemic compound is prepared byforming an aqueous solution of dothiepin hydrochloride andmono-3,6-anhydrocyclo-maltoheptaose containing 5 mmole/l ofhydrochloride and 10 mmole/l of the cyclodextrin derivative. Thesolution is then examined by nuclear magnetic resonance spectrometry at500 MHz and 298K. The spectrum obtained under these conditions is shownin FIGS. 1 and 2.

FIG. 1 shows the complete spectrum, whilst FIG. 2 shows on a largerscale that part of the spectrum of FIG. 1 corresponding to the signalsof the two enantiomers.

In FIG. 2, it is possible to see that there is a good separation of thesignals of the vinyl protons, which reaches 0.2 ppm, whereas thisseparation is only 0.03 ppm on using β-cyclodextrin under the sameconditions.

Thus, the cyclodextrin derivatives according to the invention can beused as a chiral reagent for establishing the degree of purity of anenantiomer.

We claim:
 1. A process for the preparation of amono-3,6-anhydrocyclodextrin of formula: ##STR7## in which n is 5, 6 or7, comprising reacting a monotosyl cyclodextrin of formula: ##STR8## inwhich R is a tosyl group and n is 5, 6 or 7, with lithia in an aqueousmedium.
 2. The process according to claim 1, further comprisingseparating said mono-3,6-anhydrocyclodextrin from said medium byprecipitation.
 3. The process according to claim 1, wherein n is
 6. 4. Aprocess for solubilizing a hydrophobic chemical compound in an aqueousmedium, comprising combining the hydrophobic chemical compound with amono-3,6-anhydrocyclodextrin of formula: ##STR9## in which n is 5, 6 or7, to form a water-soluble inclusion complex.
 5. An inclusion complex ofa mono-3,6-anhydrocyclodextrin of formula: ##STR10## in which n is 5, 6or 7, with a hydrophobic chemical compound.
 6. The complex according toclaim 5, wherein n is
 5. 7. The complex according to claim 5, wherein nis
 6. 8. The complex according to claim 5, wherein n is
 7. 9. Thecomplex according to claim 5, wherein the hydrophobic chemical compoundis a pharmaceutically active molecule.
 10. The complex according toclaim 9, wherein said pharmaceutically active molecule is prednisolone.11. A pharmaceutical composition, comprising the inclusion complexaccording to claim 5 and a pharmaceutically acceptable vehicle.
 12. Thepharmaceutical composition according to claim 11, wherein saidhydrophobic chemical compound is a pharmaceutically active molecule. 13.A pharmaceutical composition according to claim 12, wherein saidpharmaceutically active molecule is prednisolone.
 14. A process forcontrolling the purity of an enantiomer of an organic compound,comprising combining said enantiomer with a mono-3,6-anhydrocyclodextrinof formula: ##STR11## in which n is 5, 6 or 7, to form an inclusioncomplex of said enantiomer and subjecting said inclusion complexobtained to nuclear magnetic resonance spectrometry for detecting thepresence or absence of a different enantiomer.
 15. The process accordingto claim 14, wherein n is
 6. 16. The process according to claim 14,wherein said organic compound is dothiepin.
 17. A process for theseparation of isomers by chromatography, comprising circulating amixture of isomers in a column filled with a solid chromatographicsupport to which is covalently fixed a mono-3,6-anhydrocyclodextrin offormula (I): ##STR12## in which n is 5, 6 or 7 and collecting separatedisomers from said column.